Harnessing T cells to destroy human tumors is an attractive strategy that has met with only limited success. Among the reasons for this failure is that CD4+ T cells can differentiate to discrete subsets, one of which - CD4+ Treg cells - potently suppresses the CD4+ and CD8+ effector T cells. Although elevated percentages of Treg cells have been detected in many types of cancer, the origin of this subset of T cells in tumor microenvironment is poorly understood. Clearly, a better understanding of the origin and mechanisms of generation of CD4+ subsets would accelerate efforts to develop more effective cancer immunotherapy. The long-term goals of this proposal are to determine how Treg cells from CD4+ T cells and how Toll like receptor (TLR) signaling controls Treg cell function, and then to develop new strategies that might overcome immune suppression mediated by Treg cells, thus tipping the balance toward strong antitumor immunity. The central hypothesis of this application that tumor-reactive CD4+ T cells may be an important source of antigen-specific Treg cells generated after chronic tumor antigen stimulation is firmly grounded in the advances made during successful completion of R01 CA90327. New preliminary studies that includes the establishment of numerous CD4+ T cell lines/clones and hTLR8 transgenic mouse model, and the recent demonstration of CD4+ Treg cell functional reversal through TLR signaling, place us in an ideal position to undertake studies that will not only test this hypothesis, but will extend its predictions to possible immunotherapeutic applications as well. Specific Aim 1 seeks to identify the origin and factors contributing to the generation of CD4+ Treg cells from effector/memory Th cells and to evaluate their suppressive function in vitro and in vivo. Aim 2 will identify the signaling pathways and small molecule compounds that can be exploited to modulate Treg cell function. Ultimately in Aim 3, fundamental information regarding antigenic peptide stimulation of tumor-specific T cells and optimal methods of blocking Treg cell suppressive function will be incorporated into new cancer vaccine strategies and test in hTLR8 transgenic animal models. Successful completion of the proposed studies will provide new insights into the origin and regulation of CD4+ T cell subsets, especially Treg cells, and demonstrate the feasibility of boosting antitumor immunity by inhibiting the immunosuppressive function of antigen-specific Treg cells.